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Prime editing is a new CRISPR-based, genome-editing technology that relies on the prime editor (PE), a fusion protein of Cas9-nickase and M-MLV reverse transcriptase (RT), and a prime editing guide RNA (pegRNA) that serves both to target PE to the desired genomic locus and to carry the edit to be introduced. Here, we make advancements to the RT moiety to improve prime editing efficiencies and truncations to mitigate issues with adeno-associated virus (AAV) viral vector size limitations, which currently do not support efficient delivery of the large prime editing components. These efforts include RT variant screening, codon optimization, and PE truncation by removal of the RNase H domain and further trimming. This led to a codon-optimized and size-minimized PE that has an expression advantage (1.4-fold) and size advantage (621 bp shorter). In addition, we optimize the split intein PE system and identify Rma-based Cas9 split sites (573-574 and 673-674) that combined with the truncated PE delivered by dual AAVs result in superior AAV titer and prime editing efficiency. We also show that this minimized PE gives rise to superior lentiviral vector titers (46-fold) over the regular PE in an all-in-one PE lentiviral vector. We finally deliver the minimized PE to mouse liver by dual AAV8 vectors and show up to 6% precise editing of the PCSK9 gene, thereby demonstrating the value of this truncated split PE system for in vivo applications.

Delivery of genes to mouse liver is routinely accomplished by tail-vein injections of viral vectors or naked plasmid DNA. While viral vectors are typically injected in a low-pressure and -volume fashion, uptake of naked plasmid DNA to hepatocytes is facilitated by high pressure and volumes, also known as hydrodynamic delivery. In this study, we compare the efficacy and specificity of delivery of vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped lentiviral vectors to mouse liver by a number of injection schemes. Exploiting in vivo bioluminescence imaging as a readout after lentiviral gene transfer, we compare delivery by (1) “conventional” tail-vein injections, (2) “primed” injections, (3) “hydrodynamic” injections, or (4) direct “intrahepatic” injections into exposed livers. Reporter gene activity demonstrate potent and targeted delivery to liver by hydrodynamic injections. Enhanced efficacy is confirmed by analysis of liver sections from mice treated with GFP-encoding vectors, demonstrating 10-fold higher transduction rates and gene delivery to ∼80% of hepatocytes after hydrodynamic vector delivery. In summary, lentiviral vector transfer to mouse liver can be strongly augmented by hydrodynamic tail-vein injections, resulting in both reduced off-target delivery and transduction of the majority of hepatocytes. Our findings pave the way for more effective use of lentiviral gene delivery in the mouse. Keywords: lentivirus, high-pressure tail-vein injection, hydrodynamic delivery, in vivo, liver gene therapy, gene transfer, hepatocytes

A rapidly growing understanding of the complex circuitry of microRNA (miRNA)-mediated gene regulation is attracting attention to miRNAs as new drug targets. Targeted miRNA suppression is achieved in a sequence-specific manner by antisense RNA "decoy" molecules. Such synthetic miRNA inhibitors have reached the clinic with remarkable pace and may soon appear as new therapeutic modalities in several diseases. Shortcomings, however, include high production costs, the requirement for repeated administration, and difficulty achieving tissue-specific delivery. With the many recent landmark achievements in clinical gene therapy, new and refined vector-encoded miRNA suppression technologies are attractive for many applications, not least as tools in innumerable daily studies of miRNA biology in laboratories worldwide. Here, we provide an overview of the strategies that have been used to adapt vector-encoded inhibitors for miRNA suppression and discuss advantages related to spatiotemporal and long-term miRNA attenuation. With the remarkable new discovery of miRNA management by naturally occurring circular RNAs, RNA circles generated by trans-splicing mechanisms may prove to be well-suited carriers of decoy-type miRNA inhibitors. The community will aspire to combine circles with high-affinity miRNA decoy methodologies, and such "vectorized" RNA circles may represent new solid ways to deliver miRNA inhibitors, perhaps even with therapeutic applications.

Cutaneous homeostasis and innate immunity is procured by a complex circuitry of intercellular cytokine signaling. MicroRNAs are important posttranscriptional regulators of keratinocyte gene expression and assist in modulating the fine balance between cell proliferation and differentiation in skin. A characteristic microRNA profile in inflammatory skin suggests putative functions of microRNAs in perturbed cytokine production and signaling during chronic inflammatory skin conditions such as psoriasis. It remains unclear, however, why certain microRNAs are aberrantly expressed during skin inflammation and if they serve pro- and/or anti-inflammatory functions. In this report, we focus on cytokine regulation by microRNA-203 (miR-203), which is highly abundant in keratinocytes and upregulated in psoriatic lesions. By screening a panel of cytokines that are upregulated in psoriatic skin for regulation by miR-203, we identify the genes encoding the pro-inflammatory cytokines TNFα and IL24 as direct targets of miR-203. Studies of miR-203 overexpression, inhibition, and mutagenesis validate posttranscriptional regulation of TNFα and IL24 by miR-203 in cell lines and primary keratinocytes. Our findings suggest that miR-203 serves to fine-tune cytokine signaling and may dampen skin immune responses by repressing key pro-inflammatory cytokines.

It has been previously shown that integrase-defective HIV-1-based gene vectors can serve, with moderate efficiency, as substrate for DNA transposition by a transiently expressed Sleeping Beauty (SB) transposase. Here, we describe the enhanced gene transfer properties of a HIV-1/SB hybrid vector that allows efficient DNA transposition, facilitated by the hyperactive SB100X transposase, from vector DNA intermediates in primary human cells. Potent transposase-dependent integration of genetic cargo carried by the hybrid HIV-1/SB vector (up to 160-fold above background) is reported in human cell lines as well as in primary human fibroblasts and keratinocytes. The efficiency of transgene integration in context of the newly developed hybrid vector is comparable with that of conventional lentiviral vectors (LVs). Integration profiles of integrating HIV-1-derived vectors and SB transposons mobilized from LVs are investigated by deep sequencing of a large number of integration sites. A significant bias of lentiviral integrations in genes is reported, confirming that biological properties of the viral integration machinery facilitate preferred insertion into actively transcribed genomic regions. In sharp contrast, lentiviral insertions catalyzed by the SB100X transposase are far more random with respect to genes. Based on these properties, HIV-1/SB vectors may become valuable tools for genetic engineering and therapeutic gene transfer.

Udgivelsesdato: 2009-Jun-30 Tumor necrosis factor-alpha (TNF-alpha) is upregulated in psoriatic skin and represents a prominent target in psoriasis treatment. The level of TNF-alpha-encoding mRNA, however, is not increased in psoriatic skin, and it remains unclear whether intervention strategies based on RNA interference (RNAi) are therapeutically relevant. To test this hypothesis the present study describes first the in vitro functional screening of a panel of short hairpin RNAs (shRNAs) targeting human TNF-alpha mRNA and, next, the transfer of the most potent TNF-alpha shRNA variant, as assessed in vitro, to human skin in the psoriasis xenograft transplantation model by the use of lentiviral vectors. TNF-alpha shRNA treatment leads to amelioration of the psoriasis phentotype in the model, as documented by reduced epidermal thickness, normalization of the skin morphology, and reduced levels of TNF-alpha mRNA as detected in skin biopsies 3 weeks after a single vector injection of lentiviral vectors encoding TNF-alpha shRNA. Our data show efficient lentiviral gene delivery to psoriatic skin and therapeutic applicability of anti-TNF-alpha shRNAs in human skin. These findings validate TNF-alpha mRNA as a target molecule for a potential persistent RNA-based treatment of psoriasis and establish the use of small RNA effectors as a novel platform for target validation in psoriasis and other skin disorders.Molecular Therapy (2009); doi:10.1038/mt.2009.141.

Udgivelsesdato: 2008-Nov-4 The Sleeping Beauty (SB) transposon system represents an important alternative to viral integrating vector systems but may, as its viral counterparts, be subject to transcriptional silencing. To investigate shielding of SB-delivered transgene cassettes against transcriptional repression, we establish silencing assays in which SB vector-containing F9 murine teratocarcinoma cell clones are identified by strategies that include or exclude selection for transgene expression. Among clones carrying one or more SB transposon vectors, more than one-third are immediately silenced, and most of the remaining clones move toward silencing during prolonged passage. In line with the lack of an intrinsic ability of SB to resist silencing, we show that the stable transfection rate of SB vectors in F9 cells is significantly improved by flanking the transgene with heterologous 5'-HS4 chicken beta-globin (cHS4) insulators. In approaches based on drug selection and subsequent flow-cytometric detection of transgene expression, clones containing cHS4-insulated vectors are to a much higher degree protected against transcriptional silencing, resulting in long-term expression of the fluorescent marker. Our findings demonstrate that SB vectors, prone for transcriptional silencing by positional effects in F9 cells, are protected by insulators. We believe that insulated SB-derived vectors will become useful tools in transposon-based transgenesis and therapeutic gene transfer.Molecular Therapy (2008); doi:10.1038/mt.2008.224.

Transposon-based vectors represent promising new tools for chromosomal transgene insertion and establishment of persistent gene expression in vivo. Here, we report the development of helper-independent transposon-transposase (HITT) vectors, which contain on single plasmids (i) a Sleeping Beauty (SB) transposon containing the transgene and (ii) a SB transposase expression cassette. To obtain an optimal level of transposase expression from HITT vectors, we determined the relative strength of a panel of different promoters in mouse liver and used these promoters to drive transposase expression from injected HITT vectors carrying a human alpha(1)-antitrypsin (hAAT) expression cassette flanked by transposon inverted repeats. By correlating promoter strength with stabilized serum hAAT levels, a narrow expression window supporting high-level transposition in the liver was defined. Peak levels of long-term gene expression were obtained with promoters 30- to 40-fold less active than CMV in mouse liver, whereas reduced stable levels of hAAT were detected with both weaker and stronger promoters. Injected HITT vectors induced transposase-dependent insertion of transposon DNA into the genome of at least 5-6% of transfected hepatocytes, generating levels of persistent hAAT expression that were 2- to 4-fold higher than with an optimized two-plasmid approach. In addition, we show that HITT vectors carrying a human factor IX (hFIX)-containing transposon support (i) long-term hFIX expression in normal mice and (ii) partial phenotypic correction in a mouse model of hemophilia B. SB-based HITT vectors represent a major advance in the establishment of persistent transgene expression from nonviral gene delivery systems and should prove useful for gene transfer to tissues or cell types in which transfection efficiencies are low.

Retroviruses harbour a diploid genome of two plus-strand RNAs linked non-covalently at the dimer linkage structure. Co-packaging of two parental RNAs is a prerequisite for recombination in retroviruses, but formation of heterodimers has not been demonstrated directly in vivo. Here, we explore elements in Harvey sarcoma virus (HaSV) RNA involved in homodimerization and heterodimerization with RNA of Moloney (Mo) and Akv murine leukemia viruses (MLV). By an in vitro assay, we found that HaSV dimerization specificity could be modulated by mutations in a decanucleotide palindrome (Pal) probably folded into a kissing-loop. Autocomplementary and non-autocomplementary sequences introduced into the putative loop directed the specificity towards formation of homodimers and heterodimers, respectively. Two stem-loop (SL) structures, both exposing a GACG tetraloop, enhanced the formation of stable HaSV dimers.A similar decanucleotide palindrome has been implicated in homodimerization of MLVs. Heterodimers between HaSV RNA and Mo- or Akv MLV were unstable, but could be stabilized by introduction of two point mutations in the putative HaSV kissing-loop, creating exact complementarity with Mo/Akv MLV palindromes. Moreover, such changes increased the HaSV RNA affinity for the two MLV RNAs. Similar to HaSV RNA homodimers, formation of heterodimers with Mo- or Akv MLV RNAs was induced by the presence of GACG loops. On the basis of these results, we propose that palindromic sequences act as variable determinants of specificity and GACG tetraloops as conserved determinants in the formation of homodimers and heterodimers of gamma-retrovirus retroviral RNAs in vivo. The complementarity of loop sequences in the packaging signal upstream of the GACG tetraloops might therefore determine homo- and heterodimerization specificity and recombination activity of these viruses.

Therapeutic use of site-directed endonucleases relies on safe and effective cellular delivery, preferentially resulting in short-term enzymatic activity. Based on the packaging of Gag/GagPol-fused heterologous proteins into VSV-G-pseudotyped lentivirus-derived particles, we have established lentiviral protein transduction for delivery of DNA transposases and custom-made endonucleases. Up to 24% of targeted CCR5 and AAVS1 alleles were disrupted in primary cells, including normal human dermal fibroblasts and primary keratinocytes, exposed to lentiviral particles loaded with zinc-finger nucleases (ZFNs). By exposing human 293 cells to ‘all-in-one’ integrase-defective lentiviral vectors (IDLVs) containing a complete gene repair kit consisting of ZFNs and viral RNA carrying the donor sequence for homology-directed repair, correction of genomic mutations was obtained in more than 8% of treated cells. As shown by confocal microscopy, ZFN proteins were abundant within transduced cells one hour after initial virus exposure, but were short-lived and gone after 24 hours. In accordance, under conditions supporting comparable CCR5 indel rates, disruption of the nearby CCR2 off-target site was reduced by lentiviral delivery of ZFNs targeting CCR5 relative to a conventional transfection-based approach. As biased and uncontrolled integration into genes remains a key challenge for gene therapies based on lentiviral vector technologies, we engineered ZFN-loaded IDLVs with the capacity to insert transgenes into the human CCR5 and AAVS1 loci by a homology-driven mechanism. Targeted gene integration into safe genomic loci was observed in human cell lines (85% of analyzed clones) and in human stem cells, including CD34+ hematopoietic progenitors and induced pluripotent stem cells (iPSCs). Notably, targeted transgene insertion into safe harbors was identified in all of 23 analyzed iPSC clones. Altogether, our findings generate a new platform for targeted genome engineering based on lentiviral delivery of complete gene repair or gene insertion kits.

The PiggyBac (PB) transposon system is a potent nonviral gene delivery tool with relevance in both gene therapy and cell reprogramming for production of induced pluripotent stem cells (iPSCs). Moreover, by taking advantage of the ability of the PB transposase to excise transposon-embedded gene cassettes from the genome without leaving footprints, the PB system is unique in facilitating seamless genome editing. The need for intracellular production of the transposase, however, raises concerns related to delivery and to cytotoxicity caused by sustained transposase expression and insertional mutagenesis. Furthermore, transposon re-integration may decrease the overall efficiency of the PB-mediated excision as well as increasing the risk of adverse secondary insertions. Based on our previous work, we present a new approach for lentivirus-based delivery of PB transposase. By fusing the hyperactive PB transposase, hyPBase, to the C-terminus of the GagPol polyprotein, we show robust incorporation and subsequent release of the transposase in matured lentiviral particles. Furthermore, in an effort to limit transposon re-integration, we engineered a hyPBase variant carrying three missense mutations. This novel hyPBase variant, hyPBaseExc+/Int−, demonstrates integration levels very close to background levels, thus limiting the risk of reintegration. Notably, the ability of hyPBaseExc+/Int− to excise transposons from plasmid-borne as well as from genomically integrated PB transposon cassettes is increased up to 6.3-fold relative to the original hyPBase transposase. By fusing the hyPBaseExc+/Int− to the C-terminus of GagPol, transposase protein can be efficiently delivered to cells by lentiviral protein transduction and, in our model system, performs seamless genomic excision of PB transposon cassettes in a copy number and dose-dependent manner, resulting in excision in up to 23.6% of virus-treated cells. Using a transposon containing the puro-deltaTK transgene cassette, we furthermore show that cells with successful excision can be enriched 17-fold by negative selection with FIAU. We believe that protein transduction of hyPBaseExc+/Int− may increase the applicability and safety of transposase-directed genomic excision in iPSCs and in hard-to-transfect cell types including hematopoietic cells.

Top of pageAbstract The Sleeping Beauty (SB) transposon represents an important vehicle for in vivo gene delivery because it can stably integrate into the genomes of mammalian cells. Although SB is one of the most efficient non-viral integrating systems described to date, all previous estimations of its integrative potential have relied on the use of antibiotic selection schemes. Based on previous studies demonstrating post-integrative gene silencing with other gene transfer systems (e.g. retroviruses), we investigated how genomic integration influenced transposon expression in human cells. To do this, we devised a novel strategy to monitor SB integration irrespective of transgene expression. We first constructed single plasmids (pT/RSV- YFP.CMV-SB, pT/EF1|[alpha]|-eGFP.CMV-SB, and pT/dmEF1|[alpha]|- dmGFP.CMV-SB) encoding a GFP variant-marked transposon and an externally-located transposase expression cassette and then transfected each of these plasmids into HeLa cells. Shortly thereafter, cells were single-cell sorted by FACS and then grown in the absence of antibiotic selection to generate a large number of clonal cell lines, each of which was screened via Southern blot analysis for transposon integrations. Results indicate that in the absence of selection, the Sleeping Beauty transposition system has a true integration efficiency of 41-52%, a value that is significantly higher than the 3-10% estimates previously generated using antibiotic selection-based approaches. In addition, we find that, irrespective of promoter usage or CpG content, many transposon copies undergo post-integrational gene silencing during a 12 week time period, with silencing in 13-18% of single-integration cell lines. Furthermore, upon continued passage of the pT/RSV-YFP.CMV-SB-derived lines, the frequency of transposon silencing had increased substantially from 18% to 71% after a 42 week time period. Studies utilizing the methylation- sensitive restriction enzyme McrBC indicate that DNA methylation contributes substantially to transgene repression in the vast majority of integrated transposon copies, a finding which was further substantiated through experimental application of the DNA methyltransferase inhibitor, 5-aza-2|[prime]|-deoxycytidine. Histone deacetylation was also found to play a role in transgene repression using the histone deacetylases inhibitor trichostatin A. Overall, these transposon clones are proving invaluable in helping unravel the molecular mechanism(s) governing transcriptional gene silencing, and may someday provide the insight needed to design an appropriate integrating vector system that is capable of maintaining long-term gene expression regardless of its integration site.

DNA transposons, incorporated in nonviral or viral gene delivery systems, represent promising new tools for chromosomal transgene insertion and establishment of persistent gene expression in vivo. In a nonviral gene therapy approach based on a single plasmid vector containing (i) a transgene-tagged Sleeping Beauty (SB) transposon and (ii) a SB-derived transposase we have previously demonstrated efficient transposition and therapeutic gene expression in mouse liver (Mikkelsen et al., Mol. Ther. 2003, 8:654). Although this vector technology holds great promise as a nonviral therapeutic tool, incorporation of the transposon integration machinery into viral vector systems may be required for some applications. We have previously shown that circular DNA serves as an efficient substrate for SB transposition, whereas linear DNA only supports limited transposition. Therefore, to create a gutless 'all-in-one' adenoviral vector containing all components required for transposition we have generated adenoviral vector constructs from which plasmid-like circular DNA carrying a transgene-marked transposon and a transposase expression cassette can be released by Flp-recombinase-based recombination. To reduce possible premature DNA circle formation during vector preparation, we fused Flpe with a modified human progesterone receptor (hPR) domain that facilitates specific binding to and activation by the synthetic steroid RU486. By co-transfections with a |[beta]|-gal reporter plasmid, the Flpe-hPR protein was found to retain 20 to 50% of the wildtype Flpe activity (depending on cell line) in the presence of RU486 and was induced 6- to 44-fold by addition of RU486. Next, by using plasmid vectors containing both (i) a neo-tagged transposon and (ii) a Flpe-activated transposase expression cassette we found that the level of transposition in Flpe-hPR-transfected HeLa cells was 5-fold higher than the background level in the presence of RU486 and only slightly reduced (1.4-fold) compared to cells transfected with wildtype Flpe plasmid. We have successfully produced 'all-in-one' adenoviral vectors carrying the transposase coding sequence, the Flpe-hPR recombinase gene, and a human factor IX-marked transposon and are currently studying RU486-induced generation of circular substrates for SB-mediated transposition in in vitro-transduced cells. In summary, this study demonstrates a new approach for drug-inducible SB transposition and its use in a novel integrating adenoviral vector system with potential applications in gene therapy approaches which require long-term transgene expression.

The Sleeping Beauty (SB) transposon represents an important vehicle for in vivo gene delivery because it can stably integrate into the genomes of mammalian cells. Although SB is the most efficient non-viral integrating system described to date, all previous estimations of SB's integrative potential have relied on the use of antibiotic selection schemes. Previous work in other integrating systems (e.g. retroviruses) has demonstrated significant post-integrative gene silencing, leading us to investigate how genomic integration influences transposon expression in human cells. To do this we devised a new strategy to monitor SB integration that is not dependent on transgene expression. We constructed a single plasmid (pT/RSV-YFP.CMV-SB) containing both a YFP-marked transposon and a separate transposase expression cassette (external to the transposon), then transfected the plasmid into HeLa cells. Shortly thereafter, cells were single-cell sorted by FACS to generate approximately 100 clonal cell lines. After 8 weeks of growth (>50 cell divisions) 30% of the cell lines continue to show stable transgene expression, as determined by fluorescence microscopy and FACS analyses. We have screened these cell lines for the presence of transposon sequences by both PCR and Southern blot analyses, using various restriction enzymes to determine (i) the copy number and (ii) the molecular forms of the transposable element. Results indicate approximately 50% of the total cell lines contain an integrated transposon sequence(s), of which roughly 20% show little or no YFP expression. Our early results, based upon Hpa II (a methylation sensitive restriction enzyme) cleavage of genomic DNA, suggested the lack of YFP expression was not due to DNA methylation but to some other regulatory system. However, more recent data (including the use of the methylation specific McrBC enzyme) indicates that some of the integrated transposons do in fact become methylated over time. Possibly related, most individual cell lines show heterogeneous YFP levels throughout the cell population. Using aphidicolin (a cell cycle inhibitor) we determined that the varied YFP expression is not cell cycle dependent. We are continuing to explore post-integrative gene expression/silencing using a variety of approaches including a detailed analysis of how CpG concentration within the transposon expression cassette affects long-term gene expression. Overall, our transposon clones are proving to be an important new reagent to investigate the mechanism(s) by which integrating genetic elements (e.g. transposons and retroviruses) are silenced by the host cell, and should further our understanding of transcriptional silencing of integrating gene transfer vectors.

The Sleeping Beauty (SB) transposon system mediates stable genomic integration by a cut-and-paste transposition process and has shown tremendous success as a therapeutic vehicle for in vivo gene delivery. Nevertheless, the overall rate of transposition with SB is still insufficient for many clinical applications. Herein, we generated 141 missense mutants for the SB transposase and screened each for altered gene transfer activity in human cells using a genetic assay. We show that despite equivalent steady-state levels of wild-type (WT) and mutant transposases in transfected cells, many mutations introduced into the N-terminal DNA-binding domain (n=10) and C-terminal catalytic core domain (n=6) resulted in 2- to 4-fold higher transpositional efficiencies compared to WT. In order to better understand the transposition reaction for potential future development, we compared the functional activities of these hyperactive recombinases to that of the WT transposase using a combination of PCR and EMSAs. Results of these studies identified a number of pathways that contribute to improved transposase activity, including an altered affinity of the transposase for transposon ends, transposase conformational changes, enhanced donor cleavage activity, and increased rates of strand transfer. Importantly, many of these hyperactive SB (HSB) mutations functioned synergistically and, when combined with a hyperactive transposon, elevated transposition by 14-fold compared to the first-generation transposon system. Moreover, although WT and mutant transposases were negatively regulated at high enzyme concentrations, the HSB variants supported ~10-fold higher transpositional activity at all experimental doses, suggesting that lower doses could be administered to further improve the safety profile for SB in vivo. In addition, the use of HSB instead of the WT transposase dramatically improved the integration frequency of larger-sized elements, including ones greater than 14 kb in length. These HSB mutants should greatly extend the carrying capacity for SB, thus overcoming one of this system's major limitations. Finally, we studied the long-term in vivo persistence of two transposon reporters, β-galactosidase and human coagulation factor IX (hFIX), in the livers of adult mice following a single administration of WT- or hyperactive transposase-expressing plasmids. Results of these analyses demonstrated that these mutated enzymes could also greatly enhance SB's gene transfer capabilities in vivo, supporting stable integration in an estimated 57% of transfected mouse hepatocytes, compared to ~8% for the WT. This level of transposition was sufficient to maintain ~7-fold higher serum hFIX levels (955 ng/ml ± 241 ng/ml) than achieved in SB-expressing animals (140 ng/ml ± 56 ng/ml) for a period of 4 months (length of study). This level of hFIX obtained with the HSB mutant is ~19% of normal human levels and is well within a curative range capable of converting a severely affected hemophilia B patient to one with a much milder phenotype. Therefore, our work demonstrates the potential for improved in vivo gene delivery through directed transposase evolution, and provides a number of important insights into Sleeping Beauty transposon biology that could be exploited for future development.

The Sleeping Beauty (SB) transposon represents an important vehicle for in vivo gene delivery because it can stably integrate into the genomes of mammalian cells. Although SB is the most efficient non-viral integrating system described to date, all previous estimations of SB's integrative potential have relied on the use of antibiotic selection schemes. Previous work in other integrating systems (e.g. retroviruses) has demonstrated significant post-integrative gene silencing, leading us to investigate how genomic integration influences transposon expression in human cells. To do this we devised a new strategy to monitor SB integration that is not dependent on transgene expression. We constructed a single autonomously integrating plasmid (pT/YFP-SB) containing a YFP-marked transposon and an external plasmid-encoded transposase expression cassette, and transfected it into HeLa cells. Shortly thereafter, cells were single-cell sorted by FACS to generate a total of 183 clonal cell populations. Unexpectedly, after 6 weeks of growth (> 30 cell divisions) 28% of the cell lines continue to show stable transgene expression, as determined by fluorescence microscopy. We have begun screening these cell lines for the presence of transposon sequences by both PCR and Southern blot analyses, using various restriction enzymes to determine (i) the copy number and (ii) the molecular forms of the transposable element. Interestingly, preliminary results indicate that up to 15% of the total cell lines contain non-expressing integrated transgenes and we are currently investigating whether this may be due to post-integrative gene silencing. Surprisingly, up to 40% of the expressing cell lines contain persisting (non-dividing) episomal plasmid, as determined by DpnI/Southern blot analyses. Consequently, we are investigating whether co-delivery of SB and transposon sequences can facilitate episomal persistence in mammalian cells, possibly through transposase-mediated nuclear retention. Also of note, many individual cell lines showed heterogeneous YFP levels throughout the cell population. Currently, we are testing whether expression from an integrated transposon varies with the cell cycle, as has been seen in other integrating systems. The generation of stably silenced transposon clones provides an important new reagent to investigate the mechanism(s) by which integrating genetic elements (e.g. transposons and retroviruses) are silenced by the host cell, and should further our understanding of Sleeping Beauty biology.

The life span of patients with Fanconi Anemia (FA) is severely shortened by bone marrow failure and malignancies. Currently, the only efficient therapy is bone-marrow transplantation from a matched sibling donor. Therefore, FA is considered a good candidate disease for hematopoietic gene therapy. Our previous preclinical gene therapy experiments with Fancc knock-out mice have shown that wild-type hematopoietic stem cells (introduced by transplantation or generated by gene transfer) have a strong selective growth advantage in vivo. However, despite the successful modeling of FA gene therapy in the mouse, application of the same approach to humans is complicated by the requirement for ex vivo manipulation and scarcity of HSCs in FA patients. Consequently, the development of new gene transfer methods that support stable integration of therapeutic transgenes in vivo are greatly desired. Here, we have utilized the Sleeping Beauty (SB) transposon system to facilitate stable, transposase-mediated gene transfer of the human FANCC cDNA in vivo from a plasmid-based system. The transposase and FANCC transposon plasmid constructs were co-transfected into bone-marrow cells of Fancc mutant mice by two different methods: 1) via electroporation into enriched hematopoietic cells; 2) via direct injection into the femur cavity. In each case, the presence of the transgene was followed in treated animals by semi-quantitative PCR. Although no transgene was detectable in peripheral blood DNA 2 weeks after injection, 2/4 mice injected into the femur cavity were PCR positive 4 weeks after injection, even in the absence of selective pressure. At this time, a single low dose of cyclophosphamide (CPA) was given to provide a selective advantage for FANCC corrected cells. Four weeks after CPA administration, all the experimental electroporated animals (N=2) and femur injected animals (N=8) were strongly PCR positive in their peripheral blood and have remained stably positive for at least 9 months. Furthermore, upon transplantation of bone marrow from these mice into wild-type lethally irradiated recipients, we observed that 81/110 (74%) of day 12 CFU-S colonies were transgene positive. Similarly, 64/110 (64%) of the 12-day CFU-S colonies were transgene positive from the femur-injected donors, thus confirming that transgene integration had occurred in bona fide stem cells. Collectively, these studies demonstrate that a) hematopoietic stem cells from Fancc mutant mice are proficient in taking up plasmid DNA; b) stable integration of plasmid DNA into the genome of hematopoietic stem cells is possible and c) the efficiency of stable gene transfer using these nonviral approaches compares favorably to that achieved by alternative viral-based methods. We are in the process of analyzing transposon host cellular DNA junctions to determine the mechanism of transgene persistence in each experimental group. The above experiments have been repeated independently with more animals: N=5 for electroporated and N=6 for femur injected. Details of all these results will be discussed.